The present invention is directed to growth-state specific probes and to a method for distinguishing physiological status of bacterial cells through use of such probes, more particularly the growth state of individual bacterial cells. The present invention is especially useful for distinguishing growing from dormant or viable but nonculturable bacterial cells.
The publications and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Civilization has long recognized the relationship between fecal contamination and the outbreak of communicable disease. Two categories of bacteria routinely tested as indicative of fecal contamination are Escherichia coli and total coliforms. The Environmental Protection Agency (EPA) requires monitoring of coliform content in wastewater effluent prior to its release into recreational waters. Most U.S. communities are located in close proximity to such waterways. The EPA requires that discharged municipal effluent contain no more than 4,000 fecal coliforms per liter (Eaton et al., 1995). To meet these requirements, fecal coliform content usually is adjusted by chlorination with chlorine gas or chloramines followed by residual chlorine neutralization with sulfur dioxide (Fed. Regist. 60:62562). Since wastewater comprises a diverse community of microbial taxa, standard procedures for fecal coliform enumeration rely on selective enrichment techniques using detergent additives (Eaton et al., 1995). However, studies on coliform regrowth in chlorinated drinking water indicate that such techniques significantly underestimate coliform death due to chlorine injury that induces a viable but nonculturable xe2x80x9cVNCxe2x80x9d state. (Camper and McFeters, 1979; McFeters et al., 1986; McFeters, 1990). Because resuscitation of injured cells can occur upon nutrient resupplement, it is well recognized that most standard procedures may underestimate the incidence of the indicator species and therefore distort water quality estimates (Dawes and Penrose, 1978; Postgate and Hunter, 1962; Xu et al., 1982).
Many factors which limit bacterial proliferation can precipitate the VNC or dormant state (Morita, 1997; Oliver, 1993). Recent data indicates that VNC forms remain potentially pathogenic. Dormancy has been characterized in great detail in Vibrio (Rockabrand et al., 1995; Whitesides and Oliver, 1997) and is of particular importance in estimating the occurrence of cholera, a water-borne disease (Colwell, 1996). In natural samples, the disparity between total and culturable cell counts and the diversity of 16S rRNA sequences apparent in uncultivated samples compared to culture collections, indicate that most bacteria are unculturable (Staley and Konopka, 1985). This suggests that dormancy is widespread. Despite efforts to clarify the physiological basis for this state, the relationship between dormancy and culturability has remained unclear. In contrast, much has been learned about the early stationary phase which precedes dormancy (Blum, 1997; Goodrich-Blair et al., 1996; Hengge-Aronis, 1996). Current understanding and strategies for the analysis of the VNC state are limited. Thus, there is a definite need to understand the VNC state and determine the physiological status and the relationship between dormancy and culturability and for development of new methodologies capable of detecting microorganisms in the VNC state.
Utilization of bacteria in industrial settings has become common. For example, microbial-based hazardous waste treatment processes (bioremediation) have been applied to the treatment of numerous hazardous wastes and associated liquids including: refinery and petrochemical production oily sludges and asphaltic type wastes, process waste slurries from organic chemicals production and soils contaminated with fuel oils. Bioremediation involves exploiting abilities of indigenous or augmented microorganisms to metabolize organic susbstrates. Bioremediation systems are designed to achieve optimal conditions for microbial degradation. To this end, it is desirable to prevent bacterial growth limitations. The prevention of bacterial growth limitations requires accurate assessment and monitoring of the physiology and growth state of the bacteria.
Availability of recombinant bacteria have facilitated the industrial production of various enzymes and other proteins of therapeutic value. Escherichia coli is the most frequently used prokaryotic expression system for the production of heterologous proteins, including recombinant antibodies. Proteins made in E. coli have been widely used in detection, imaging, diagnosis, and therapy. In order to increase fermenter productivity, cell density in culture in excess of 100 g dry weight/L is desirable; however, cells at higher densities are difficult to maintain and the cells are more likely to die. Therefore, careful monitoring of growth state is necessary to assure productivity.
Assessment of the progress of treatment of human and animal diseases with antimicrobial agents relies on conventional culturability measurements. Assessment based on detection of only culturable cells may overestimate success of treatment due to the presence of dormant bacteria. Thus, it is desirable to establish new methodologies capable of detecting bacterial growth state for use in a variety of applications, including: treatment of wastewater, industrial fermentations, bioremediation and treatment of bacterial disease. The present invention solves this need as illustrated herein.
Utilization of bacteria in industrial settings has become common. For example, microbial-based hazardous waste treatment processes (bioremediation) have been applied to the treatment of numerous hazardous wastes and associated liquids including: refinery and petrochemical production oily sludges and asphaltic type wastes, process waste slurries from organic chemicals production and soils contaminated with fuel oils. Bioremediation involves exploiting abilities of indigenous or augmented microorganisms to metabolize organic substrates. Bioremediation systems are designed to achieve optimal conditions for microbial degradation. To this end, it is desirable to prevent bacterial growth limitations. The prevention of bacterial growth limitations requires accurate assessment and monitoring of the physiology and growth state of the bacteria.